Bordetella bronchiseptica is an etiologic agent of infectious tracheobronchitis (Kennel Cough) in dogs, atrophic rhinitis in pigs, and respiratory infections in rabbits and guinea pigs. Bordetella infections depress the respiratory clearance mechanisms (mucociliary elevator), facilitating invasion by other organisms. Colonization of the upper respiratory tract by the bacteria occurs via firm attachment to the ciliated respiratory epithelium, followed by rapid proliferation and an inflammatory response. Binding to host tissue is the primary step in the establishment of infection, and B. bronchispetica produce an array of moleculesthat mediate attachment, including filamentous hemagglutinin, tracheal cytotoxin, pertactin, and fimbriae. Several genes coding for fimbrial subunits have been previously reported, including fim 2, fim 3, fim A, and fim X. Most of these fimbrae have counterparts in B. pertussis. 
Two fimbrial phenotypes of B. bronchiseptica have been defined serologically as fim 2 and fim 3. They are reactive with factor specific antiserums historically used for serotyping B.pertussis isolates. Fim A and fim X (Boschwitz, J. S., H. G. J. van der Heide, F. R. Mooi, and D. A. Relman [1997] J. Bacteriol. 179:7882-7885), two other major fimbrial proteins, are also expressed in B.bronchiseptica; however, their antigenic characteristics are not known. Existence of other bordetella fimbriae has been hypothesized (Mooi, F. R., W. H. Jansen, H. Brunings, H. Gielen, H. G. J van der Heide, H. C. Walvoort, and Guinee, P. A. M. [1992] Micro. Pathog. 12:127-135).
The expression of bordetella fimbrial genes is positively regulated through the bvg locus (Willems, R., A. Paul, H. G. J. van der Heide, A. R. ter Avest, and F. R. Mooi [1990] EMBO J. 9:2803-2809) and their promoter regions have in common a stretch of cytosines referred to as a C-stretch (Willems et al. [1990] supra; Riboli, B., P. Pedroni, A. Cuzzoni, and F. de Ferra [1991] Micro. Pathog. 10:393-403), and two motifs 5xe2x80x2 to the C-stretch comprising an ABR or fim box (Riboli et al., [1991] supra). Characterization of fimbrial genes and their products is important to the understanding of fimbrial gene regulation, for serological diagnosis, for identification of potential subunit vaccine candidates, and to understand mechanisms of cellular adhesion.
The subject invention concerns novel polynucleotides of Bordetella species, and the polypeptides encoded thereby. The subject invention further provides vaccines useful for providing protection against Bordetella infections.
In a specific embodiment, the subject invention provides a unique Bordetella bronchiseptica gene. This gene was isolated from a cosmid constructed with B. bronchiseptica chromosomal DNA. This cosmid, when introduced into attachment deficient B. bronchiseptica, increases the binding of the bacteria to epithelial cells. The fimbrial gene, referred to as fim N, has been subcloned and sequenced. The predicted protein from this gene has about 59% and about 52% homology with B. bronchiseptica fim 2 and fim 3, respectively. Fim N encodes a protein predicted to be similar in size and structure to those encoded by fim 2 and fim 3. Serologically, the fim N protein appears to have cross-reactivity with serotype 2 antibodies. The fim N protein is present in B. bronchiseptica isolates associated with disease in several species of veterinary importance.
Various abbreviations are found within this specification. Their terms and meanings are as follows: aa, amino acid(s); Ab, antibody(ies); Ap, ampicillin; ELISA, enzyme-linked immunosorbent assay; EtdBr, ethidium bromide; IPTG, isopropyl xcex2-D-thiogalactopyranoside; kb, kilobase; LB, Luria-Bertani (medium); mAB, monoclonal Ab; Nixe2x80x94-NTA, nickel-nitrilotriacetic acid; nt, nucleotide(s); O.D., optical density; oligo, oligonucleotide; ORF, open reading frame; p, plasmid; PCR, polymerase chain reaction; PBS, phosphate buffered saline; PBSTW, PBS containing polyoxyethylenesorbitan monolaurate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tc, tetracycline.